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1.
Nature ; 626(7999): 574-582, 2024 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-38086421

RESUMEN

The intrinsic mechanisms that regulate neurotoxic versus neuroprotective astrocyte phenotypes and their effects on central nervous system degeneration and repair remain poorly understood. Here we show that injured white matter astrocytes differentiate into two distinct C3-positive and C3-negative reactive populations, previously simplified as neurotoxic (A1) and neuroprotective (A2)1,2, which can be further subdivided into unique subpopulations defined by proliferation and differential gene expression signatures. We find the balance of neurotoxic versus neuroprotective astrocytes is regulated by discrete pools of compartmented cyclic adenosine monophosphate derived from soluble adenylyl cyclase and show that proliferating neuroprotective astrocytes inhibit microglial activation and downstream neurotoxic astrocyte differentiation to promote retinal ganglion cell survival. Finally, we report a new, therapeutically tractable viral vector to specifically target optic nerve head astrocytes and show that raising nuclear or depleting cytoplasmic cyclic AMP in reactive astrocytes inhibits deleterious microglial or macrophage cell activation and promotes retinal ganglion cell survival after optic nerve injury. Thus, soluble adenylyl cyclase and compartmented, nuclear- and cytoplasmic-localized cyclic adenosine monophosphate in reactive astrocytes act as a molecular switch for neuroprotective astrocyte reactivity that can be targeted to inhibit microglial activation and neurotoxic astrocyte differentiation to therapeutic effect. These data expand on and define new reactive astrocyte subtypes and represent a step towards the development of gliotherapeutics for the treatment of glaucoma and other optic neuropathies.


Asunto(s)
Astrocitos , Neuroprotección , Adenilil Ciclasas/metabolismo , Astrocitos/citología , Astrocitos/enzimología , Astrocitos/metabolismo , Diferenciación Celular , Núcleo Celular/metabolismo , Supervivencia Celular , AMP Cíclico/metabolismo , Citoplasma/metabolismo , Macrófagos/metabolismo , Macrófagos/patología , Microglía/metabolismo , Microglía/patología , Traumatismos del Nervio Óptico/metabolismo , Traumatismos del Nervio Óptico/patología , Traumatismos del Nervio Óptico/terapia , Células Ganglionares de la Retina/citología , Células Ganglionares de la Retina/metabolismo , Sustancia Blanca/metabolismo , Sustancia Blanca/patología , Glaucoma/patología , Glaucoma/terapia
2.
Nature ; 595(7867): 444-449, 2021 07.
Artículo en Inglés | MEDLINE | ID: mdl-34194047

RESUMEN

The size of the transcriptional program of long non-coding RNAs in the mammalian genome has engendered discussions about their biological roles1, particularly the promoter antisense (PAS) transcripts2,3. Here we report the development of an assay-referred to as chromatin isolation by RNA-Cas13a complex-to quantitatively detect the distribution of RNA in the genome. The assay revealed that PAS RNAs serve as a key gatekeeper of a broad transcriptional pause release program, based on decommissioning the 7SK small nuclear RNA-dependent inhibitory P-TEFb complex. Induction of PAS RNAs by liganded ERα led to a significant loss of H3K9me3 and the release of basally recruited HP1α and KAP1 on activated target gene promoters. This release was due to PAS RNA-dependent recruitment of H3K9me3 demethylases, which required interactions with a compact stem-loop structure in the PAS RNAs, an apparent feature of similarly regulated PAS RNAs. Activation of the ERα-bound MegaTrans enhancer, which is essential for robust pause release, required the recruitment of phosphorylated KAP1, with its transfer to the cognate promoters permitting 17ß-oestradiol-induced pause release and activation of the target gene. This study reveals a mechanism, based on RNA structure, that mediates the function of PAS RNAs in gene regulation.


Asunto(s)
Conformación de Ácido Nucleico , Regiones Promotoras Genéticas/genética , ARN sin Sentido/química , ARN sin Sentido/genética , Activación Transcripcional/genética , Línea Celular , Homólogo de la Proteína Chromobox 5/metabolismo , Proteína Sustrato Asociada a CrK , Receptor alfa de Estrógeno/metabolismo , Histonas/química , Histonas/metabolismo , Humanos , Histona Demetilasas con Dominio de Jumonji/metabolismo , Ligandos , Factor B de Elongación Transcripcional Positiva/metabolismo , ARN Polimerasa II/metabolismo , Estabilidad del ARN , Proteína 28 que Contiene Motivos Tripartito/metabolismo
3.
Mol Cell ; 69(5): 757-772.e7, 2018 03 01.
Artículo en Inglés | MEDLINE | ID: mdl-29499132

RESUMEN

As most of the mitochondrial proteome is encoded in the nucleus, mitochondrial functions critically depend on nuclear gene expression and bidirectional mito-nuclear communication. However, mitochondria-to-nucleus communication pathways in mammals are incompletely understood. Here, we identify G-Protein Pathway Suppressor 2 (GPS2) as a mediator of mitochondrial retrograde signaling and a transcriptional activator of nuclear-encoded mitochondrial genes. GPS2-regulated translocation from mitochondria to nucleus is essential for the transcriptional activation of a nuclear stress response to mitochondrial depolarization and for supporting basal mitochondrial biogenesis in differentiating adipocytes and brown adipose tissue (BAT) from mice. In the nucleus, GPS2 recruitment to target gene promoters regulates histone H3K9 demethylation and RNA POL2 activation through inhibition of Ubc13-mediated ubiquitination. These findings, together, reveal an additional layer of regulation of mitochondrial gene transcription, uncover a direct mitochondria-nuclear communication pathway, and indicate that GPS2 retrograde signaling is a key component of the mitochondrial stress response in mammals.


Asunto(s)
Núcleo Celular/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Mitocondrias/metabolismo , Biogénesis de Organelos , Transducción de Señal/fisiología , Células 3T3-L1 , Transporte Activo de Núcleo Celular/fisiología , Animales , Núcleo Celular/genética , Células HeLa , Histonas/genética , Histonas/metabolismo , Humanos , Péptidos y Proteínas de Señalización Intracelular/genética , Metilación , Ratones , Mitocondrias/genética , Regiones Promotoras Genéticas/fisiología , Activación Transcripcional/fisiología
4.
Mol Cell ; 71(4): 526-539.e8, 2018 08 16.
Artículo en Inglés | MEDLINE | ID: mdl-30118678

RESUMEN

Nuclear receptors induce both transcriptional activation and repression programs responsible for development, homeostasis, and disease. Here, we report a previously overlooked enhancer decommissioning strategy underlying a large estrogen receptor alpha (ERα)-dependent transcriptional repression program. The unexpected signature for this E2-induced program resides in indirect recruitment of ERα to a large cohort of pioneer factor basally active FOXA1-bound enhancers that lack cognate ERα DNA-binding elements. Surprisingly, these basally active estrogen-repressed (BAER) enhancers are decommissioned by ERα-dependent recruitment of the histone demethylase KDM2A, functioning independently of its demethylase activity. Rather, KDM2A tethers the E3 ubiquitin-protein ligase NEDD4 to ubiquitylate/dismiss Pol II to abrogate eRNA transcription, with consequent target gene downregulation. Thus, our data reveal that Pol II ubiquitylation/dismissal may serve as a potentially broad strategy utilized by indirectly bound nuclear receptors to abrogate large programs of pioneer factor-mediated, eRNA-producing enhancers.


Asunto(s)
Elementos de Facilitación Genéticos , Receptor alfa de Estrógeno/genética , Proteínas F-Box/genética , Factor Nuclear 3-alfa del Hepatocito/genética , Histona Demetilasas con Dominio de Jumonji/genética , Ubiquitina-Proteína Ligasas Nedd4/genética , ARN Polimerasa II/genética , Secuencia de Bases , Sitios de Unión , Sistemas CRISPR-Cas , Estradiol/farmacología , Receptor alfa de Estrógeno/metabolismo , Proteínas F-Box/metabolismo , Edición Génica/métodos , Regulación de la Expresión Génica/efectos de los fármacos , Células HEK293 , Factor Nuclear 3-alfa del Hepatocito/metabolismo , Histonas/genética , Histonas/metabolismo , Humanos , Histona Demetilasas con Dominio de Jumonji/metabolismo , Células MCF-7 , Ubiquitina-Proteína Ligasas Nedd4/metabolismo , Unión Proteica , ARN/genética , ARN/metabolismo , ARN Polimerasa II/metabolismo , Transducción de Señal , Transcripción Genética/efectos de los fármacos , Ubiquitinación/efectos de los fármacos
5.
Cell ; 139(6): 1069-83, 2009 Dec 11.
Artículo en Inglés | MEDLINE | ID: mdl-19962179

RESUMEN

Chromosomal translocations are a hallmark of leukemia/lymphoma and also appear in solid tumors, but the underlying mechanism remains elusive. By establishing a cellular model that mimics the relative frequency of authentic translocation events without proliferation selection, we report mechanisms of nuclear receptor-dependent tumor translocations. Intronic binding of liganded androgen receptor (AR) first juxtaposes translocation loci by triggering intra- and interchromosomal interactions. AR then promotes site-specific DNA double-stranded breaks (DSBs) at translocation loci by recruiting two types of enzymatic activities induced by genotoxic stress and liganded AR, including activation-induced cytidine deaminase and the LINE-1 repeat-encoded ORF2 endonuclease. These enzymes synergistically generate site-selective DSBs at juxtaposed translocation loci that are ligated by nonhomologous end joining pathway for specific translocations. Our data suggest that the confluence of two parallel pathways initiated by liganded nuclear receptor and genotoxic stress underlies nonrandom tumor translocations, which may function in many types of tumors and pathological processes.


Asunto(s)
Neoplasias de la Próstata/genética , Receptores Androgénicos/metabolismo , Transcripción Genética , Translocación Genética , Línea Celular Tumoral , Daño del ADN , Reparación del ADN , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Humanos , Intrones , Elementos de Nucleótido Esparcido Largo , Masculino , Sistemas de Lectura Abierta , Serina Endopeptidasas/genética , Serina Endopeptidasas/metabolismo , Transactivadores/genética , Transactivadores/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Regulador Transcripcional ERG
6.
J Biol Chem ; 298(4): 101674, 2022 04.
Artículo en Inglés | MEDLINE | ID: mdl-35148987

RESUMEN

Adeno-associated viruses (AAVs) targeting specific cell types are powerful tools for studying distinct cell types in the central nervous system (CNS). Cis-regulatory modules (CRMs), e.g., enhancers, are highly cell-type-specific and can be integrated into AAVs to render cell type specificity. Chromatin accessibility has been commonly used to nominate CRMs, which have then been incorporated into AAVs and tested for cell type specificity in the CNS. However, chromatin accessibility data alone cannot accurately annotate active CRMs, as many chromatin-accessible CRMs are not active and fail to drive gene expression in vivo. Using available large-scale datasets on chromatin accessibility, such as those published by the ENCODE project, here we explored strategies to increase efficiency in identifying active CRMs for AAV-based cell-type-specific labeling and manipulation. We found that prescreening of chromatin-accessible putative CRMs based on the density of cell-type-specific transcription factor binding sites (TFBSs) can significantly increase efficiency in identifying active CRMs. In addition, generation of synthetic CRMs by stitching chromatin-accessible regions flanking cell-type-specific genes can render cell type specificity in many cases. Using these straightforward strategies, we generated AAVs that can target the extensively studied interneuron and glial cell types in the retina and brain. Both strategies utilize available genomic datasets and can be employed to generate AAVs targeting specific cell types in CNS without conducting comprehensive screening and sequencing experiments, making a step forward in cell-type-specific research.


Asunto(s)
Encéfalo , Dependovirus , Retina , Coloración y Etiquetado , Factores de Transcripción , Animales , Sitios de Unión , Encéfalo/citología , Encéfalo/metabolismo , Cromatina/genética , Cromatina/metabolismo , Dependovirus/genética , Dependovirus/metabolismo , Ratones , Retina/citología , Retina/metabolismo , Coloración y Etiquetado/métodos , Factores de Transcripción/metabolismo
7.
Mol Cell ; 59(2): 188-202, 2015 Jul 16.
Artículo en Inglés | MEDLINE | ID: mdl-26166704

RESUMEN

Enhancers instruct spatio-temporally specific gene expression in a manner tightly linked to higher-order chromatin architecture. Critical chromatin architectural regulators condensin I and condensin II play non-redundant roles controlling mitotic chromosomes. But the chromosomal locations of condensins and their functional roles in interphase are poorly understood. Here we report that both condensin complexes exhibit an unexpected, dramatic estrogen-induced recruitment to estrogen receptor α (ER-α)-bound eRNA(+) active enhancers in interphase breast cancer cells, exhibiting non-canonical interaction with ER-α via its DNA-binding domain (DBD). Condensins positively regulate ligand-dependent enhancer activation at least in part by recruiting an E3 ubiquitin ligase, HECTD1, to modulate the binding of enhancer-associated coactivators/corepressors, including p300 and RIP140, permitting full eRNA transcription, formation of enhancer:promoter looping, and the resultant coding gene activation. Collectively, our results reveal an important, unanticipated transcriptional role of interphase condensins in modulating estrogen-regulated enhancer activation and coding gene transcriptional program.


Asunto(s)
Adenosina Trifosfatasas/metabolismo , Proteínas de Unión al ADN/metabolismo , Elementos de Facilitación Genéticos , Receptor alfa de Estrógeno/metabolismo , Complejos Multiproteicos/metabolismo , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Adenosina Trifosfatasas/antagonistas & inhibidores , Adenosina Trifosfatasas/genética , Secuencia de Bases , Sitios de Unión , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Cromatina/genética , Cromatina/metabolismo , ADN de Neoplasias/genética , Proteínas de Unión al ADN/antagonistas & inhibidores , Proteínas de Unión al ADN/genética , Estradiol/metabolismo , Femenino , Técnicas de Silenciamiento del Gen , Humanos , Interfase , Células MCF-7 , Modelos Biológicos , Datos de Secuencia Molecular , Complejos Multiproteicos/antagonistas & inhibidores , Complejos Multiproteicos/genética , Proteínas Nucleares/metabolismo , Proteína de Interacción con Receptores Nucleares 1 , Regiones Promotoras Genéticas , Unión Proteica , ARN Neoplásico/genética , ARN Neoplásico/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitinación
8.
Mol Cell ; 46(1): 91-104, 2012 Apr 13.
Artículo en Inglés | MEDLINE | ID: mdl-22424771

RESUMEN

The association between hyperinflammatory states and numerous diseases is widely recognized, but our understanding of the molecular strategies that have evolved to prevent uncontrolled activation of inflammatory responses remains incomplete. Here, we report a critical, nontranscriptional role of GPS2 as a guardian against hyperstimulation of the TNF-α-induced gene program. GPS2 cytoplasmic actions are required to specifically modulate RIP1 ubiquitylation and JNK activation by inhibiting TRAF2/Ubc13 enzymatic activity. In vivo relevance of GPS2 anti-inflammatory role is confirmed by inhibition of TNF-α target genes in macrophages and by improved insulin signaling in the adipose tissue of aP2-GPS2 transgenic mice. As the nontranscriptional role is complemented by GPS2 functioning as positive and negative cofactor for nuclear receptors, in vivo overexpression also results in elevated circulating level of Resistin and development of hepatic steatosis. Together, these studies define GPS2 as a molecular guardian required for precise control of inflammatory responses involved in immunity and homeostasis.


Asunto(s)
Tejido Adiposo/metabolismo , Homeostasis , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Macrófagos/metabolismo , Tejido Adiposo/inmunología , Animales , Línea Celular , Proteínas Activadoras de GTPasa/genética , Proteínas Activadoras de GTPasa/inmunología , Proteínas Activadoras de GTPasa/metabolismo , Inflamación/genética , Inflamación/inmunología , Inflamación/metabolismo , Insulina/genética , Insulina/inmunología , Insulina/metabolismo , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/inmunología , MAP Quinasa Quinasa 4/genética , MAP Quinasa Quinasa 4/inmunología , MAP Quinasa Quinasa 4/metabolismo , Macrófagos/inmunología , Ratones , Ratones Transgénicos , Resistina/genética , Resistina/inmunología , Resistina/metabolismo , Transducción de Señal/genética , Transducción de Señal/inmunología , Factor de Necrosis Tumoral alfa/genética , Factor de Necrosis Tumoral alfa/inmunología , Factor de Necrosis Tumoral alfa/metabolismo , Enzimas Ubiquitina-Conjugadoras/genética , Enzimas Ubiquitina-Conjugadoras/inmunología , Enzimas Ubiquitina-Conjugadoras/metabolismo , Ubiquitinación/genética , Ubiquitinación/inmunología
9.
Nature ; 500(7464): 598-602, 2013 Aug 29.
Artículo en Inglés | MEDLINE | ID: mdl-23945587

RESUMEN

Although recent studies have indicated roles of long non-coding RNAs (lncRNAs) in physiological aspects of cell-type determination and tissue homeostasis, their potential involvement in regulated gene transcription programs remains rather poorly understood. The androgen receptor regulates a large repertoire of genes central to the identity and behaviour of prostate cancer cells, and functions in a ligand-independent fashion in many prostate cancers when they become hormone refractory after initial androgen deprivation therapy. Here we report that two lncRNAs highly overexpressed in aggressive prostate cancer, PRNCR1 (also known as PCAT8) and PCGEM1, bind successively to the androgen receptor and strongly enhance both ligand-dependent and ligand-independent androgen-receptor-mediated gene activation programs and proliferation in prostate cancer cells. Binding of PRNCR1 to the carboxy-terminally acetylated androgen receptor on enhancers and its association with DOT1L appear to be required for recruitment of the second lncRNA, PCGEM1, to the androgen receptor amino terminus that is methylated by DOT1L. Unexpectedly, recognition of specific protein marks by PCGEM1-recruited pygopus 2 PHD domain enhances selective looping of androgen-receptor-bound enhancers to target gene promoters in these cells. In 'resistant' prostate cancer cells, these overexpressed lncRNAs can interact with, and are required for, the robust activation of both truncated and full-length androgen receptor, causing ligand-independent activation of the androgen receptor transcriptional program and cell proliferation. Conditionally expressed short hairpin RNA targeting these lncRNAs in castration-resistant prostate cancer cell lines strongly suppressed tumour xenograft growth in vivo. Together, these results indicate that these overexpressed lncRNAs can potentially serve as a required component of castration-resistance in prostatic tumours.


Asunto(s)
ARN Largo no Codificante/genética , Receptores Androgénicos/metabolismo , Activación Transcripcional/genética , Regulación hacia Arriba/genética , Animales , Castración , Línea Celular Tumoral , Proliferación Celular , Elementos de Facilitación Genéticos/genética , Humanos , Masculino , Ratones , Ratones Desnudos , Trasplante de Neoplasias , Regiones Promotoras Genéticas/genética , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/patología , Factores de Transcripción/metabolismo
10.
Nature ; 498(7455): 516-20, 2013 Jun 27.
Artículo en Inglés | MEDLINE | ID: mdl-23728302

RESUMEN

The functional importance of gene enhancers in regulated gene expression is well established. In addition to widespread transcription of long non-coding RNAs (lncRNAs) in mammalian cells, bidirectional ncRNAs are transcribed on enhancers, and are thus referred to as enhancer RNAs (eRNAs). However, it has remained unclear whether these eRNAs are functional or merely a reflection of enhancer activation. Here we report that in human breast cancer cells 17ß-oestradiol (E2)-bound oestrogen receptor α (ER-α) causes a global increase in eRNA transcription on enhancers adjacent to E2-upregulated coding genes. These induced eRNAs, as functional transcripts, seem to exert important roles for the observed ligand-dependent induction of target coding genes, increasing the strength of specific enhancer-promoter looping initiated by ER-α binding. Cohesin, present on many ER-α-regulated enhancers even before ligand treatment, apparently contributes to E2-dependent gene activation, at least in part by stabilizing E2/ER-α/eRNA-induced enhancer-promoter looping. Our data indicate that eRNAs are likely to have important functions in many regulated programs of gene transcription.


Asunto(s)
Elementos de Facilitación Genéticos/genética , Estrógenos/farmacología , ARN no Traducido/genética , Activación Transcripcional/efectos de los fármacos , Proteínas de Ciclo Celular/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , Estradiol/farmacología , Receptor alfa de Estrógeno/metabolismo , Humanos , Ligandos , Células MCF-7 , Conformación de Ácido Nucleico/efectos de los fármacos , Regiones Promotoras Genéticas/genética , ARN no Traducido/biosíntesis , ARN no Traducido/metabolismo , Transcripción Genética/efectos de los fármacos , Transcripción Genética/genética , Activación Transcripcional/genética , Cohesinas
11.
Nucleic Acids Res ; 45(9): e71, 2017 May 19.
Artículo en Inglés | MEDLINE | ID: mdl-28108660

RESUMEN

Experimental evidence indicates that about 60% of miRNA-binding activity does not follow the canonical rule about the seed matching between miRNA and target mRNAs, but rather a non-canonical miRNA targeting activity outside the seed or with a seed-like motifs. Here, we propose a new unbiased method to identify canonical and non-canonical miRNA-binding sites from peaks identified by Ago2 Cross-Linked ImmunoPrecipitation associated to high-throughput sequencing (CLIP-seq). Since the quality of peaks is of pivotal importance for the final output of the proposed method, we provide a comprehensive benchmarking of four peak detection programs, namely CIMS, PIPE-CLIP, Piranha and Pyicoclip, on four publicly available Ago2-HITS-CLIP datasets and one unpublished in-house Ago2-dataset in stem cells. We measured the sensitivity, the specificity and the position accuracy toward miRNA binding sites identification, and the agreement with TargetScan. Secondly, we developed a new pipeline, called miRBShunter, to identify canonical and non-canonical miRNA-binding sites based on de novo motif identification from Ago2 peaks and prediction of miRNA::RNA heteroduplexes. miRBShunter was tested and experimentally validated on the in-house Ago2-dataset and on an Ago2-PAR-CLIP dataset in human stem cells. Overall, we provide guidelines to choose a suitable peak detection program and a new method for miRNA-target identification.


Asunto(s)
Secuenciación de Nucleótidos de Alto Rendimiento , MicroARNs/metabolismo , Secuencias de Aminoácidos , Proteínas Argonautas/química , Proteínas Argonautas/genética , Benchmarking , Sitios de Unión , Humanos , MicroARNs/química , Conformación de Ácido Nucleico , Sensibilidad y Especificidad , Programas Informáticos
12.
J Biol Chem ; 292(7): 2754-2772, 2017 02 17.
Artículo en Inglés | MEDLINE | ID: mdl-28039360

RESUMEN

Non-proteolytic ubiquitin signaling mediated by Lys63 ubiquitin chains plays a critical role in multiple pathways that are key to the development and activation of immune cells. Our previous work indicates that GPS2 (G-protein Pathway Suppressor 2) is a multifunctional protein regulating TNFα signaling and lipid metabolism in the adipose tissue through modulation of Lys63 ubiquitination events. However, the full extent of GPS2-mediated regulation of ubiquitination and the underlying molecular mechanisms are unknown. Here, we report that GPS2 is required for restricting the activation of TLR and BCR signaling pathways and the AKT/FOXO1 pathway in immune cells based on direct inhibition of Ubc13 enzymatic activity. Relevance of this regulatory strategy is confirmed in vivo by B cell-targeted deletion of GPS2, resulting in developmental defects at multiple stages of B cell differentiation. Together, these findings reveal that GPS2 genomic and non-genomic functions are critical for the development and cellular homeostasis of B cells.


Asunto(s)
Linfocitos B/citología , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Enzimas Ubiquitina-Conjugadoras/antagonistas & inhibidores , Animales , Células de la Médula Ósea/citología , Diferenciación Celular , Perfilación de la Expresión Génica , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Receptores de Antígenos de Linfocitos B/metabolismo , Transducción de Señal , Ubiquitinación
13.
Proc Natl Acad Sci U S A ; 112(5): 1380-5, 2015 Feb 03.
Artículo en Inglés | MEDLINE | ID: mdl-25605944

RESUMEN

Substantial evidence supports the hypothesis that enhancers are critical regulators of cell-type determination, orchestrating both positive and negative transcriptional programs; however, the basic mechanisms by which enhancers orchestrate interactions with cognate promoters during activation and repression events remain incompletely understood. Here we report the required actions of LIM domain-binding protein 1 (LDB1)/cofactor of LIM homeodomain protein 2/nuclear LIM interactor, interacting with the enhancer-binding protein achaete-scute complex homolog 1, to mediate looping to target gene promoters and target gene regulation in corticotrope cells. LDB1-mediated enhancer:promoter looping appears to be required for both activation and repression of these target genes. Although LDB1-dependent activated genes are regulated at the level of transcriptional initiation, the LDB1-dependent repressed transcription units appear to be regulated primarily at the level of promoter pausing, with LDB1 regulating recruitment of metastasis-associated 1 family, member 2, a component of the nucleosome remodeling deacetylase complex, on these negative enhancers, required for the repressive enhancer function. These results indicate that LDB1-dependent looping events can deliver repressive cargo to cognate promoters to mediate promoter pausing events in a pituitary cell type.


Asunto(s)
Corticotrofos/fisiología , Proteínas de Unión al ADN/fisiología , Elementos de Facilitación Genéticos , Proteínas con Dominio LIM/fisiología , Regiones Promotoras Genéticas , Animales , Línea Celular , Proteínas de Unión al ADN/metabolismo , Proteínas con Dominio LIM/metabolismo , Ratones , Ratones Noqueados
14.
Nature ; 470(7333): 264-8, 2011 Feb 10.
Artículo en Inglés | MEDLINE | ID: mdl-21307941

RESUMEN

Genome-wide association studies have identified single nucleotide polymorphisms (SNPs) in the 9p21 gene desert associated with coronary artery disease (CAD) and type 2 diabetes. Despite evidence for a role of the associated interval in neighbouring gene regulation, the biological underpinnings of these genetic associations with CAD or type 2 diabetes have not yet been explained. Here we identify 33 enhancers in 9p21; the interval is the second densest gene desert for predicted enhancers and six times denser than the whole genome (P < 6.55 × 10(-33)). The CAD risk alleles of SNPs rs10811656 and rs10757278 are located in one of these enhancers and disrupt a binding site for STAT1. Lymphoblastoid cell lines homozygous for the CAD risk haplotype show no binding of STAT1, and in lymphoblastoid cell lines homozygous for the CAD non-risk haplotype, binding of STAT1 inhibits CDKN2BAS (also known as CDKN2B-AS1) expression, which is reversed by short interfering RNA knockdown of STAT1. Using a new, open-ended approach to detect long-distance interactions, we find that in human vascular endothelial cells the enhancer interval containing the CAD locus physically interacts with the CDKN2A/B locus, the MTAP gene and an interval downstream of IFNA21. In human vascular endothelial cells, interferon-γ activation strongly affects the structure of the chromatin and the transcriptional regulation in the 9p21 locus, including STAT1-binding, long-range enhancer interactions and altered expression of neighbouring genes. Our findings establish a link between CAD genetic susceptibility and the response to inflammatory signalling in a vascular cell type and thus demonstrate the utility of genome-wide association study findings in directing studies to novel genomic loci and biological processes important for disease aetiology.


Asunto(s)
Cromosomas Humanos Par 9/genética , Enfermedad de la Arteria Coronaria/genética , Elementos de Facilitación Genéticos/genética , Predisposición Genética a la Enfermedad/genética , Variación Genética , Interferón gamma/farmacología , Transducción de Señal/efectos de los fármacos , Alelos , Línea Celular , Cromatina/efectos de los fármacos , Cromatina/genética , Cromatina/metabolismo , Secuencia Conservada/genética , Inhibidor p15 de las Quinasas Dependientes de la Ciclina/genética , Diabetes Mellitus Tipo 2/genética , Células Endoteliales/efectos de los fármacos , Células Endoteliales/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/genética , Técnicas de Silenciamiento del Gen , Estudio de Asociación del Genoma Completo , Haplotipos/genética , Células HeLa , Humanos , Interferón-alfa/genética , Desequilibrio de Ligamiento , Masculino , Polimorfismo de Nucleótido Simple/genética , Unión Proteica/efectos de los fármacos , Purina-Nucleósido Fosforilasa/genética , Factor de Transcripción STAT1/biosíntesis , Factor de Transcripción STAT1/deficiencia , Factor de Transcripción STAT1/genética , Factor de Transcripción STAT1/metabolismo , Población Blanca/genética
15.
Proc Natl Acad Sci U S A ; 111(25): 9235-40, 2014 Jun 24.
Artículo en Inglés | MEDLINE | ID: mdl-24928520

RESUMEN

Understanding the mechanisms by which compounds discovered using cell-based phenotypic screening strategies might exert their effects would be highly augmented by new approaches exploring their potential interactions with the genome. For example, altered androgen receptor (AR) transcriptional programs, including castration resistance and subsequent chromosomal translocations, play key roles in prostate cancer pathological progression, making the quest for identification of new therapeutic agents and an understanding of their actions a continued priority. Here we report an approach that has permitted us to uncover the sites and mechanisms of action of a drug, referred to as "SD70," initially identified by phenotypic screening for inhibitors of ligand and genotoxic stress-induced translocations in prostate cancer cells. Based on synthesis of a derivatized form of SD70 that permits its application for a ChIP-sequencing-like approach, referred to as "Chem-seq," we were next able to efficiently map the genome-wide binding locations of this small molecule, revealing that it largely colocalized with AR on regulatory enhancers. Based on these observations, we performed the appropriate global analyses to ascertain that SD70 inhibits the androgen-dependent AR program, and prostate cancer cell growth, acting, at least in part, by functionally inhibiting the Jumonji domain-containing demethylase, KDM4C. Global location of candidate drugs represents a powerful strategy for new drug development by mapping genome-wide location of small molecules, a powerful adjunct to contemporary drug development strategies.


Asunto(s)
Antagonistas de Receptores Androgénicos/farmacología , Sistemas de Liberación de Medicamentos/métodos , Proteínas de Neoplasias/genética , Neoplasias de la Próstata/genética , Receptores Androgénicos/genética , Ensayos Antitumor por Modelo de Xenoinjerto/métodos , Animales , Antineoplásicos , Línea Celular Tumoral , Análisis Mutacional de ADN/métodos , Humanos , Masculino , Ratones , Proteínas de Neoplasias/metabolismo , Neoplasias de la Próstata/tratamiento farmacológico , Neoplasias de la Próstata/metabolismo , Receptores Androgénicos/metabolismo , Translocación Genética
16.
Nature ; 466(7305): 508-12, 2010 Jul 22.
Artículo en Inglés | MEDLINE | ID: mdl-20622854

RESUMEN

While reversible histone modifications are linked to an ever-expanding range of biological functions, the demethylases for histone H4 lysine 20 and their potential regulatory roles remain unknown. Here we report that the PHD and Jumonji C (JmjC) domain-containing protein, PHF8, while using multiple substrates, including H3K9me1/2 and H3K27me2, also functions as an H4K20me1 demethylase. PHF8 is recruited to promoters by its PHD domain based on interaction with H3K4me2/3 and controls G1-S transition in conjunction with E2F1, HCF-1 (also known as HCFC1) and SET1A (also known as SETD1A), at least in part, by removing the repressive H4K20me1 mark from a subset of E2F1-regulated gene promoters. Phosphorylation-dependent PHF8 dismissal from chromatin in prophase is apparently required for the accumulation of H4K20me1 during early mitosis, which might represent a component of the condensin II loading process. Accordingly, the HEAT repeat clusters in two non-structural maintenance of chromosomes (SMC) condensin II subunits, N-CAPD3 and N-CAPG2 (also known as NCAPD3 and NCAPG2, respectively), are capable of recognizing H4K20me1, and ChIP-Seq analysis demonstrates a significant overlap of condensin II and H4K20me1 sites in mitotic HeLa cells. Thus, the identification and characterization of an H4K20me1 demethylase, PHF8, has revealed an intimate link between this enzyme and two distinct events in cell cycle progression.


Asunto(s)
Ciclo Celular/fisiología , Proteínas Cromosómicas no Histona/metabolismo , Histona Demetilasas/metabolismo , Histonas/metabolismo , Lisina/metabolismo , Factores de Transcripción/metabolismo , Adenosina Trifosfatasas/química , Adenosina Trifosfatasas/metabolismo , Línea Celular , Cromatina/metabolismo , Proteínas Cromosómicas no Histona/química , Proteínas Cromosómicas no Histona/deficiencia , Proteínas Cromosómicas no Histona/genética , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/metabolismo , Células HeLa , Histona Demetilasas/química , Histona Demetilasas/genética , N-Metiltransferasa de Histona-Lisina/metabolismo , Histonas/química , Factor C1 de la Célula Huésped/genética , Factor C1 de la Célula Huésped/metabolismo , Humanos , Metilación , Complejos Multiproteicos/química , Complejos Multiproteicos/metabolismo , Fosforilación , Regiones Promotoras Genéticas , Estructura Terciaria de Proteína , Factores de Transcripción/química , Factores de Transcripción/deficiencia , Factores de Transcripción/genética
17.
iScience ; 27(6): 110100, 2024 Jun 21.
Artículo en Inglés | MEDLINE | ID: mdl-38947520

RESUMEN

Retinal ganglion cell (RGC) differentiation is tightly controlled by extrinsic and intrinsic factors. Growth and differentiation factor 15 (GDF-15) promotes RGC differentiation, opposite to GDF-11 which inhibits RGC differentiation, both in the mouse retina and in human stem cells. To deepen our understanding of how these two closely related molecules confer opposing effects on retinal development, here we assess the transcriptional profiles of mouse retinal progenitors exposed to exogenous GDF-11 or -15. We find a dichotomous effect of GDF-15 on RGC differentiation, decreasing RGCs expressing residual pro-proliferative genes and increasing RGCs expressing non-proliferative genes, suggestive of greater RGC maturation. Furthermore, GDF-11 promoted the differentiation of photoreceptors and amacrine cells. These data enhance our understanding of the mechanisms underlying the differentiation of RGCs and photoreceptors from retinal progenitors and suggest new approaches to the optimization of protocols for the differentiation of these cell types.

18.
Nature ; 441(7093): 646-50, 2006 Jun 01.
Artículo en Inglés | MEDLINE | ID: mdl-16511445

RESUMEN

Precise regulation of the NFAT (nuclear factor of activated T cells) family of transcription factors (NFAT1-4) is essential for vertebrate development and function. In resting cells, NFAT proteins are heavily phosphorylated and reside in the cytoplasm; in cells exposed to stimuli that raise intracellular free Ca2+ levels, they are dephosphorylated by the calmodulin-dependent phosphatase calcineurin and translocate to the nucleus. NFAT dephosphorylation by calcineurin is countered by distinct NFAT kinases, among them casein kinase 1 (CK1) and glycogen synthase kinase 3 (GSK3). Here we have used a genome-wide RNA interference (RNAi) screen in Drosophila to identify additional regulators of the signalling pathway leading from Ca2+-calcineurin to NFAT. This screen was successful because the pathways regulating NFAT subcellular localization (Ca2+ influx, Ca2+-calmodulin-calcineurin signalling and NFAT kinases) are conserved across species, even though Ca2+-regulated NFAT proteins are not themselves represented in invertebrates. Using the screen, we have identified DYRKs (dual-specificity tyrosine-phosphorylation regulated kinases) as novel regulators of NFAT. DYRK1A and DYRK2 counter calcineurin-mediated dephosphorylation of NFAT1 by directly phosphorylating the conserved serine-proline repeat 3 (SP-3) motif of the NFAT regulatory domain, thus priming further phosphorylation of the SP-2 and serine-rich region 1 (SRR-1) motifs by GSK3 and CK1, respectively. Thus, genetic screening in Drosophila can be successfully applied to cross evolutionary boundaries and identify new regulators of a transcription factor that is expressed only in vertebrates.


Asunto(s)
Drosophila/enzimología , Drosophila/genética , Genoma de los Insectos/genética , Factores de Transcripción NFATC/metabolismo , Proteínas Serina-Treonina Quinasas/clasificación , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Tirosina Quinasas/clasificación , Proteínas Tirosina Quinasas/metabolismo , Interferencia de ARN , Animales , Quinasa de la Caseína I/metabolismo , Drosophila/metabolismo , Genómica , Glucógeno Sintasa Quinasa 3/metabolismo , Interleucina-2/genética , Factores de Transcripción NFATC/química , Fosforilación , Proteínas Serina-Treonina Quinasas/deficiencia , Proteínas Serina-Treonina Quinasas/genética , Estructura Terciaria de Proteína , Proteínas Tirosina Quinasas/deficiencia , Proteínas Tirosina Quinasas/genética , Transcripción Genética/genética , Quinasas DyrK
19.
Nature ; 441(7090): 179-85, 2006 May 11.
Artículo en Inglés | MEDLINE | ID: mdl-16582901

RESUMEN

Antigen stimulation of immune cells triggers Ca2+ entry through Ca2+ release-activated Ca2+ (CRAC) channels, promoting the immune response to pathogens by activating the transcription factor NFAT. We have previously shown that cells from patients with one form of hereditary severe combined immune deficiency (SCID) syndrome are defective in store-operated Ca2+ entry and CRAC channel function. Here we identify the genetic defect in these patients, using a combination of two unbiased genome-wide approaches: a modified linkage analysis with single-nucleotide polymorphism arrays, and a Drosophila RNA interference screen designed to identify regulators of store-operated Ca2+ entry and NFAT nuclear import. Both approaches converged on a novel protein that we call Orai1, which contains four putative transmembrane segments. The SCID patients are homozygous for a single missense mutation in ORAI1, and expression of wild-type Orai1 in SCID T cells restores store-operated Ca2+ influx and the CRAC current (I(CRAC)). We propose that Orai1 is an essential component or regulator of the CRAC channel complex.


Asunto(s)
Canales de Calcio/metabolismo , Proteínas de Drosophila/metabolismo , Proteínas de la Membrana/metabolismo , Mutación/genética , Inmunodeficiencia Combinada Grave/genética , Inmunodeficiencia Combinada Grave/metabolismo , Animales , Transporte Biológico , Calcio/metabolismo , Portador Sano , Cromosomas Humanos Par 12/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Drosophila melanogaster/inmunología , Conductividad Eléctrica , Dosificación de Gen , Genoma Humano , Heterocigoto , Humanos , Escala de Lod , Proteínas de la Membrana/genética , Factores de Transcripción NFATC/metabolismo , Proteína ORAI1 , Fenotipo , Polimorfismo de Nucleótido Simple/genética , Interferencia de ARN , Inmunodeficiencia Combinada Grave/inmunología , Linfocitos T/inmunología , Linfocitos T/metabolismo
20.
Stem Cell Reports ; 17(12): 2690-2703, 2022 12 13.
Artículo en Inglés | MEDLINE | ID: mdl-36368332

RESUMEN

Retinal ganglion cell (RGC) replacement therapy could restore vision in glaucoma and other optic neuropathies. We developed a rapid protocol for directly induced RGC (iRGC) differentiation from human stem cells, leveraging overexpression of NGN2. Neuronal morphology and neurite growth were observed within 1 week of induction; characteristic RGC-specific gene expression confirmed identity. Calcium imaging demonstrated γ-aminobutyric acid (GABA)-induced excitation characteristic of immature RGCs. Single-cell RNA sequencing showed more similarities between iRGCs and early-stage fetal human RGCs than retinal organoid-derived RGCs. Intravitreally transplanted iRGCs survived and migrated into host retinas independent of prior optic nerve trauma, but iRGCs protected host RGCs from neurodegeneration. These data demonstrate rapid iRGC generation in vitro into an immature cell with high similarity to human fetal RGCs and capacity for retinal integration after transplantation and neuroprotective function after optic nerve injury. The simplicity of this system may benefit translational studies on human RGCs.


Asunto(s)
Glaucoma , Traumatismos del Nervio Óptico , Humanos , Células Ganglionares de la Retina , Traumatismos del Nervio Óptico/metabolismo , Retina , Células Madre
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